Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by loss of motor neurons. Approximately 10% of familial ALS cases are caused by mutations in superoxide dismutase (SOD). Although expression of disease-linked mutant SOD in transgenic mice has led to the development of successful mouse models of the disorder, invertebrate models amenable to genetic analysis have not yet been described. To enable a comprehensive genetic analysis of the mechanisms underlying motor neuron death in ALS, we have developed a genetic model in Drosophila melanogaster. Expression of disease-associated mutant human SOD proteins in transgenic flies produces progressive locomotor dysfunction that ends in paralysis and truncates lifespan. Motor neurons in transgenic animals show degenerative changes and develop intracytoplasmic inclusions containing SOD. Transgenic animals expressing normal human SOD have no locomotor dysfunction or motor neuron degeneration. These features replicate key manifestations of ALS in patients. We now propose to exploit the genetic potential of the system by generating second site suppressors and enhancers of mutant SOD-induced neurodegeneration. A sensitive and robust flight assay will be used to select de novo modifiers in a forward genetic screen. Modifiers of neurodegeneration will be characterized molecularly. The effects of modifiers on locomotor function will be characterized, as will the ability of modifiers to influence motor neuron degeneration and inclusion formation. Mammalian homologues of these Drosophila modifiers will be familial ALS gene candidates and likely components of mammalian neurodegenerative pathways. We will also test the role of candidate pathways, including protein aggregation, oxidative stress and glutamate excitotoxicity in mutant SOD-induced neurodegeneration. SOD-containing protein aggregates will be characterized at the biochemical level and colocalization of ubiquitin and heat shock proteins to inclusions evaluated. Genetic manipulation of heat shock proteins and the ubiquitin/proteasome system will be performed. The presence of oxidative damage will be assayed by immunohistochemical analysis. Genetic studies will evaluate the functional importance of oxidative damage to motor neuron degeneration. The role of excitotoxicity will be explored with genetic manipulations, in particular by overexpression of glial glutamate transporters. [unreadable] [unreadable]